β-benzyloxyaspartate derivatives with amino group on benzene ring

ABSTRACT

L-threo-β-benzyloxyaspartate derivatives having a substituent on the benzene ring, represented by the following formula (1): 
                         
wherein R is hydrogen, a linear or branched lower aliphatic acyl group with the acyl portion optionally substituted, an alicyclic acyl group, an aromatic acyl group with a substituent on the aromatic ring, an amino acid-derived group or a biotin derivative-derived group,
 
having an amino substituent on the benzene ring, and salts thereof, which can easily bind to affinity column chromatography carriers as ligands of glutamate transporter proteins.

This application is the national phase of international applicationPCT/JP02/06286 filed 24 Jun. 2002 which designated the U.S.

TECHNICAL FIELD

The present invention relates to L-glutamate uptake inhibitors, and morespecifically, it relates to derivatives of optically activeL-threo-β-benzyloxyaspartate having an amino substituent on the benzenering, represented by the following formula (1) and having activity whichinhibits uptake of glutamate by L-glutamate transporters.

wherein R is hydrogen, a linear or branched lower aliphatic acyl groupwith the acyl portion optionally substituted, an alicyclic acyl group,an aromatic acyl group with a substituent on the aromatic ring, an aminoacid-derived group or a biotin derivative-derived group.

Development of these compounds constitutes a starting point for thedevelopment of inhibitors of glutamate uptake by L-glutamatetransporters, and is expected to lead to treatment for neuropathicdisorders and neurodegenerative diseases such as epilepsy, Huntington'sdisease, amyotrophic lateral sclerosis (ALS) and Alzheimer's disease.

BACKGROUND ART

L-glutamate is a excitatory neurotransmitter found in the centralnervous system of mammals, and it is known not only to induce rapidneurotransmission between synapses but also to be involved on a higherlevel in the complex physiological processes of memory and learning.Excitatory neurotransmission between synapses begins with release ofglutamate from the presynapse, and fades with rapid glutamate uptakefrom the synaptic cleft by high-affinity glutamate transporters found innerve endings and glial cells (Attwell, D. and Nicholls, D., TIPS 68-74,1991).

Reduced sodium-dependent glutamate uptake activity in portions ofpatient brains has been reported in several genetic neurodegenerativediseases (Rothstein, J. D. et al., N. Eng. J. Med. 326, 1464-1468,1992). For this reason, activation and inhibition of glutamatetransporter function are becoming objects of focus in connection withsuch diseases.

In initial steps of research, study of glutamate transporters wascarried out primarily using synaptosomes prepared from brain tissue ormembrane specimens from the kidney and small intestine. Later, aftercloning of sodium-dependent high-affinity glutamate transporter cDNA in1992, research was conducted from a molecular biological perspective(Pines, G. et al., Nature 360, 464-467, 1992; Storck, T. et al., Proc.Natl. Acad. Sci. USA, 89, 10955-10959, 1992; Kanai, Y. et al., Nature360, 467-471, 1992). In 1994, the human glutamate transporter gene wascloned and five subtypes, EAAT1 to EAAT5, were categorized (Arriza, J.L. et al., J. Neurosci. 14, 5559-5569; Fairman, W. A. et al., Nature,375, 599-603, 1995; Arriza, J. L. et al., Proc. Natl. Acad. Sci. USA 94,4155-4160, 1997).

However, given the low homology of glutamate transporter protein withother neurotransmitter transporters and the difficulty of inferringtransmembrane regions based on hydrophobisity, there is stilldisagreement regarding the 3-dimensional structure and substraterecognition site structure (Grunewald, M. et al., Neuron 21, 623-632,1998; , Seal, R. P. et al., Neuron 25, 695-706, 2000).

In light of these circumstances, it is desirable to develop variousglutamate transporter inhibitors, and especially inhibitors thatfunction as blockers, toward elucidation of the relationship between theglutamate transporter family and neuropathic disorders andneurodegenerative diseases such as epilepsy, Huntington's disease,amyotrophic lateral sclerosis (ALS) and Alzheimer's disease.

As a result of investigation for glutamate uptake inhibitors usingsynaptosomes according to the prior art, compounds such asthreo-β-hydroxyaspartate and CCG-III[(2S,1′S,2′R)-2-(carboxycyclopropyl)glycine], t-2,4-PDC(trans-pyrrolidine-2,4-dicarboxylic acid) and the like have hithertobeen identified as glutamate uptake inhibitors, and these are themselvestaken up as substrates by transporters and thus act as inhibitors thatcompetitively inhibit glutamate uptake.

The glutamate uptake inhibitors such as kainic acid and dihydrokainicacid were demonstrated by electrophysiological studies to be blockersthat inhibit glutamate uptake without themselves being taken up. It wasfurther shown that these compounds act only on EAAT2 (GLT-1 type) of thefive EAAT subtypes (Arriza, J. L. et al., J. Neurosci. 14, 5559-5569,1994). Nevertheless, these compounds have also exhibited strongexcitatory effects on ion-channel glutamate receptors.

The present inventors have reported that β-hydroxyaspartate derivativeshaving substituents at the β-position exhibit an uptake-inhibitingeffect for all of the five EAAT subtypes (Lebrun, B. et al., J. Biol.Chem. 272, 20336-20339, 1997; Shimamoto, K. et al., Mol. Pharmacol. 53,195-201, 1998; Shigeri, Y. et al., J. Neurochem. 79, 297-302, 2001).Among them, it was found that compounds with bulky substituents at theβ-position act as blockers for all of the subtypes, inhibiting not onlyuptake of glutamate but also heteroexchange-based glutamate efflux andsodium ion influx (Chatton, J-Y. et al., Brain Res. 893, 46-52, 2001).In particular, L-threo-β-benzyloxyaspartate (L-TBOA), because of itspowerful blocking effect and its lower affinity for glutamate receptorcompared to existing inhibitors, has become a standard substance used inglutamate transporter research.

SUMMARY OF THE INVENTION

Four stereoisomers of β-hydroxyaspartate exist, and it is the L-threoform that exhibits the strongest uptake inhibition among them(Shimamoto, K. et al., Bioorg. Med. Chem. Lett. 10, 2407-2410, 2000).The conventional TBOA is used in the DL form, but it has been desired toaccomplish selective synthesis of the L-threo form in order to properlyinvestigate the structure/activity relationship.

At the same time, protein purification is essential for elucidating the3-dimensional structure of the glutamate transporter and shedding lighton the substrate transport mechanism and substrate-binding site. Andaffinity column chromatography is an effective means of proteinpurification. Protein purification using antibodies has already beenattempted, but this has been inconvenient because the strong bindingbetween the antibodies and the protein results in loss of the originalprotein function upon elution. Using a blocker as the affinity columnligand would allow elution under mild conditions, and therefore blockershaving substituents that can bind to affinity columns have also been atarget of research.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors focused on the strong affinity of TBOA, andconsidered synthesizing TBOA derivatives with a substituent on thebenzene ring and allowing the substituent to bind to the column carrier.Upon extensive research aimed at selectively synthesizingL-threo-hydroxyaspartate derivatives, we invented a synthesis pathwaywhereby desired L-threo-benzyloxyasparatate derivatives can be obtainedusing optically active epoxide as the starting material, and found thatβ-benzyloxyaspartate derivatives having an amino group on the benzenering retain uptake inhibiting activity even when substitution occurs onthe amino group.

Compounds having acyl groups as substituents function as glutamatetransporter inhibitors with inhibiting action equivalent to or exceedingthat of TBOA. In particular, compounds having a benzoyl derivative werefound to exhibit vastly increased activity and were superior to TBOA asinhibitors. On the other hand, compounds having amino acids assubstituents permit ready binding between the free amino groups andcarboxylic or halogenated alkyl groups of commercially available columncarriers. Alternatively, compounds with biotinyl groups allow bindingwith commercially available avidin columns. It was thus found that thesecompounds allow sufficient binding to column carriers while maintainingblocker activity, and the present invention was thereby completed. Inother words, the present invention provides derivatives of opticallyactive β-benzyloxyaspartate having an amino substituent on the benzenering, represented by chemical formula (1), and salts thereof, asglutamate transporter blockers and as affinity column ligands.

wherein R is hydrogen, a linear or branched lower aliphatic acyl groupwith the acyl portion optionally substituted, an alicyclic acyl group,an aromatic acyl group with a substituent on the aromatic ring, an aminoacid-derived group or a biotin derivative-derived group.

More specifically, in formula (1), R is hydrogen, a linear or branchedC₁-C₈ lower aliphatic acyl group, a C₄-C₈ alicyclic acyl group, a C₅-C₁₅aromatic acyl group, an amino acid derivative or a biotin derivative.Examples of linear or branched lower aliphatic and alicyclic acyl groupsrepresented by R include acetyl, propionyl, n-butanoyl, sec-butanoyl,n-pentanoyl, pivaloyl, phenylacetyl and cyclohexylcarbonyl. Substituentsmay also be present on the acyl group and examples of substtituentsinclude hydroxyl group, thiol group, amino group and carboxyl group.Examples of aromatic acyl groups represented by R include benzoyl,naphthoyl and pyridylcarbonyl. Substituents may also be present on thearomatic ring. Examples of substituents on the aromatic ring includelinear or branched C₁-C₇ alkyl, C₄-C₁₀ aryl, C₁-C₆ alkoxyl, nitro,cyano, amino, C₁-C₇ acylamino, carboxyl, halogen, halogenated C₁-C₆alkyl, biotinyl, and biotinylalkyl with the alkyl portion being C₁-C₆,and the like. Examples of amino acids represented by R include glycyl,alanyl, β-alanyl and cysteinyl. Examples of biotin derivativesrepresented by R include biotinyl and biotinyl-β-alanyl.

The compounds of the invention may be obtained as salts by ordinarymethods. Alkali metal salts such as sodium salts and potassium salts andalkaline earth metal salts such as calcium salts, as well as ammoniumsalts, are all included as such salts according to the invention. Saltsmay also be obtained with ordinary acids. Inorganic acid salts such ashydrochloric acid salts and sulfuric acid salts, and organic acid saltssuch as acetic acid salts, citric acid salts and trifluoroacetic acidsalts are also included as such salts according to the invention.

The position of the substituent on the benzene ring may be any one ofthe three positions of ortho-, meta- or para-, according to theinvention. However, study of the structure/activity relationship of thecompounds has revealed that in the case where an amino group ispositioned on the benzene ring, the meta-position of the amino groupprovides strongest activity. The synthesis scheme below, therefore,shows examples of introduction for obtaining compounds with substituentsat the meta-position; nevertheless, agents with different substitutionpatterns may be used for introduction of desired substituents.

The compounds of the invention may be synthesized in the followingmanner. For example, compounds wherein R is β-alanyl, biotinyl-β-alanylor propionyl-β-alanyl may be synthesized according to the followingscheme.

It is a characteristic feature of the synthesis method of the inventionthat an optically active starting material may be used for compound (1)in the above scheme. The hydroxyl-protecting group of the compound isfirst removed with an alkali metal hydroxide such as NaOH and then theresulting free hydroxyl group is reacted with a benzoylisocyanate toobtain the benzoylcarbamate represented by formula (2). As the reactionconditions, the benzoylisocyanate is added to a THF (tetrahydrofuran)solution in approximately 1.2 equivalents at room temperature, and themixture is stirred for about 30 minutes. The compound of formula (2) isreacted in the presence of a catalytic amount of tetrabutylammoniumiodide under weakly basic conditions, to produce a mixture of theL-threo cyclocarbamates represented by formula (3a) and formula (3b). Asthe reaction conditions, potassium carbonate is added at approximately 2equivalents and tetrabutylammonium iodide in approximately 0.15equivalent to an acetonitrile solution, and the mixture is stirred atroom temperature for from 14 hours to 18 hours.

This synthesis method of the invention selectively yields a L-threocyclocarbamate mixture as a production intermediate. Methods describedin prior art publications (Bioorg. Med. Chem. Lett. 10, 2407-2410, 2000)have required column purification of the threo-form from a mixturecontaining the erythro-form, but the method of the present inventionsignificantly reduces workload by dispensing with the purification.

For nitrobenzylation, compound (6) was used as the substrate instead ofthe known lactone intermediate (Bioorg. Med. Chem. Lett. 10, 2407-2410,2000), in order to suppress isomerization to the erythro-form. Whileisomerization of about 30% is seen with the conventional substrate,using the present compound results in only trace amounts, rendering it amore threo-selective production method. As the reaction conditions,sodium hydride is added at approximately 1.5 equivalents andtetrabutylammonium iodide at approximately 0.3 equivalent to a DMFsolution of compound (6) cooled to about −20° C., and then nitrobenzylbromide (preferably 3-nitrobenzyl bromide) is added at approximately 1.5equivalents and the mixture is stirred at about −20° C. for about 30minutes and then at about 0° C. for about 30 minutes. In this reaction,the yield can be greatly improved by using a nitro group as the aminoequivalent, since when a 3-protected-aminobenzyl bromide is used insteadof 3-nitrobenzyl bromide, the reaction will not proceed sufficientlyeven with a prolonged reaction time and an increased temperature.Compound (7) obtained by this reaction can then be easily converted tothe corresponding amino compound by reduction of the nitro group.Subsequent acylation by reaction with an acid chloride or carboxylicacid and a condensation agent can convert it to a compound having thedesired substituent. Compound (7) is therefore useful as an intermediatefor synthesis of the target amino-substituted benzyloxyaspartate.

EFFECT OF THE INVENTION

Well-known methods can be used to determine inhibitory activity of thecompounds of the invention on glutamate transporters. For example, thecompounds of the invention have been confirmed to inhibit uptake of¹⁴C-labeled glutamate into cells by human EAAT2 and EAAT3 stablyexpressed on MDCK (Madin-Darby Canine Kidney) cells or transientlyexpressed on COS-1 cells. This indicates that the compounds of theinvention may play a role in elucidating the glutamate transportermechanism, and are useful for research on structure/activityrelationship, protein structure analysis and the like, in connectionwith neurodegenerative diseases.

Preparation of Affinity Column

Some of the Compounds of formula (1) may be used as ligands of anaffinity column for isolation and/or purification of glutamatetransporter proteins. Well-known methods may be used to prepare anaffinity column. (For coupling reactions in general, refer to AffinityChromatography Handbook: Principles and Methods, published by AmershamPharmacia Biotech; for reactions of biotin derivatives, D. Savage, G.Matton, S. Desai, G. Nielander, S. Morgensen, E. Conklin, Avidin-BiotinChemistry: A handbook, Pierce Chemical Company (Rockford, USA), 1992.)

Purification of Protein by Affinity Column

A liquid sample suspected of containing a target protein may beintroduced into a column prepared as described above. The liquid flow issuspended to incubate the column for a certain period, e.g., about 30min. The protein is eluted with an elution buffer (e.g., 0.1 M HCl, pH2.2). If necessary, the protein solution is passed through a desaltingcolumn to remove the salt.

Some compounds of formula (1) are useful for radio-isotope labeledligands for identification of transporter proteins. Isotope labeledligands may be obtained by well known synthetic procedures, using thehydroxybenzoyl intermediate for R group in the formula (1) with thereaction, for example, of labeled methyl iodide to yield the desiredlabeled ligand as shown in Scheme 2. Some of the radio-isotope labeledmethyl iodides are commercially available, including, deuterium-labeledmethyl iodide, tritium-labeled methyl iodide, Carbon 14-labeld or Carbon11-labeled methyl iodides.

EXAMPLES

(2S,3R)-Benzoylcarbamic acid [3-(benzyloxymethyl)oxiranyl]methyl ester(2)

A 1N sodium hydroxide aqueous solution (17.5 mL, 17.5 mmol) was added toa THF solution (20 ml) containing commercially available(2S,3R)-[3-(benzyloxymethyl)oxiranyl]methanol p-nitrobenzoic acid ester(5.0 g, 14.6 mmol), and the mixture was stirred at 0° C. for one hour.The reaction solution was extracted with ether, and the organic layerwas washed with water and dried over magnesium sulfate. The solvent wasdistilled off to obtain an oily alcohol. This was dissolved in THF (20mL), and a THF solution containing benzoylisocyanate (2.57 g, 17.5 mmol)was added at room temperature. The reaction solution was stirred at roomtemperature for 30 minutes, and a saturated ammonium chloride aqueoussolution was added to quench the reaction. After ether extraction, theorganic layer was dried over magnesium sulfate. The solvent wasdistilled off and the resulting residue was purified by silica gelcolumn chromatography (ether/hexane=3/1) to obtain 5.8 g of the titlecompound (>100%). This product, though containing a small amount ofimpurity (benzamide) was used without further purification for thefollowing reaction. A portion thereof was purified by recrystallization(ether/hexane) as an analysis sample.

mp 79-81° C.; [α]_(D) −22.9° (c 0.80, CHCl₃); ¹H NMR (CDCl₃, 400 MHz);δ3.31 (m 2H), 3.64 (dd, 1H, J=6.0, 11.5 Hz), 3.73 (dd, 1H, J=4.3, 11.5Hz), 4.15 (dd, 1H, J=7.0, 12.0 Hz), 4.54 (dd, 1H, J=3.5, 12.0 Hz), 4.54(d, 1H, J=12.0 Hz), 4.61 (d, 1H, J=12.0 Hz), 7.30 (m, 1H), 7.35 (m, 4H),7.48 (ddd, 2H, J=1.0, 7.5, 7.8 Hz), 7.59 (tt, 1H, J=1.0, 7.5 Hz), 7.83(ddd, 2H, J=1.0, 1.0, 7.8 Hz), 8.33 (s, 1H).

(4R,1′S)-4-(2-Benzyloxy-1-benzoyloxyethyl)-oxazolidin-2-one (3a)

(4R,5S)-4-benzoyloxymethyl-5-benzyloxymethyl-oxazolidin-2-one (3b)

Potassium carbonate (4.04 g, 29.2 mmol) and tetrabutylammonium iodide(800 mg, 2.2 mmol) were added to an acetonitrile solution (100 mL)containing 5.8 g of benzoylcarbamate (2), and the mixture was stirred atroom temperature for 18 hours. Saturated ammonium chloride aqueoussolution was added to the reaction solution to quench the reaction.After ether extraction, the organic layer was dried over magnesiumsulfate. The solvent was distilled off and the resulting residue waspurified by silica gel column chromatography (ether/hexane=2/1) toobtain 4.87 g of a mixture of the title compounds (3 steps, 98%).

3a: mp 85-87° C.; [α]_(D) +43.6° (c 0.55, CHCl₃); ¹H NMR (CDCl₃, 400MHz); δ3.68 (d, 2H, J=5.0 Hz), 4.11 (ddd, 1H, J=, 4.5, 5.0, 5.5 Hz),4.27 (dd, 1H, J=5.5, 11.5 Hz), 4.44 (dd, 1H, J=4.5, 11.5 Hz), 4.57 (dt,1H, J=5.0, 5.0 Hz), 4.58 (d, 1H, J=11.5 Hz), 4.61 (d, 1H, J=11.5 Hz),6.08 (br s, 1H), 7.33 (m, 5H), 7.43 (ddd, 2H, J=1.0, 7.5, 7.8 Hz), 7.57(tt, 1H, J=1.0, 7.5 Hz), 8.00 (ddd, 2H, J=1.0, 1.0, 7.8 Hz). 3b: mp116-117° C.; [α]_(D) −52.6° (c 0.86, CHCl₃); ¹H NMR (CDCl₃, 400 MHz);δ3.75 (dd, 1H, J=4.5, 10.5 Hz), 3.83 (dd, 1H, J=3.8, 10.5 Hz), 4.24 (dd,1H, J=4.8, 8.8 Hz), 4.29 (ddd, 1H, J=3.5, 4.8, 8.5 Hz), 4.49 (d, 1H,J=8.5 Hz), 4.51 (d, 1H, J=8.5 Hz), 4.54 (d, 1H, J=8.8 Hz), 5.16 (ddd,1H, J=3.5, 3.8, 8.5 Hz), 5.98 (s, 1H), 7.29 (m, 5H), 7.44 (m, 2H), 7.58(tt, 1H, J=1.5, 7.5 Hz), 8.03 (m, 2H).

(2R,3S)-4-benzyloxy-2-N-tert-butoxycarbonylamino-1,3-butanediol (4)

Barium hydroxide (octahydrate) (11.3 g, 35.7 mmol) and water (100 mL)were added to an ethanol solution (100 mL) containing cyclic carbamate(3) (4.05 g, 11.9 mmol), and the suspension was stirred at 80° C. for 18hours. After cooling on ice, the pH was adjusted to 3 with 10% dilutedsulfuric acid. The precipitate was filtered off with celite, thefiltrate was concentrated, di-t-butyl-dicarbonate (5.5 mL, 23.8 mmol)and 1,4-dioxane (100 mL) were added and the pH was adjusted to 9 with a1N sodium hydroxide solution. The reaction mixture was stirred at roomtemperature for 18 hours and neutralized with 1N hydrochloric acid,after which extraction was performed with ethyl acetate and the organiclayer dried over magnesium sulfate. The residue obtained by distillingoff the solvent was purified by silica gel column chromatography(ether/hexane=3/1) to obtain 3.06 g of the title compound (2 steps,83%).

Oily: [α]_(D) +3.1° (c 0.87, CHCl₃); ¹H NMR (CDCl₃, 400 MHz); δ1.37 (s,9H), 2.88 (br s, 1H), 3.12 (s, 1H), 3.46 (dd, 1H, J=7.5, 9.5 Hz), 3.53(dd, 1H, J=4.5, 9.5 Hz), 3.66 (m, 1H), 3.69 (m, 1H), 3.79 (m, 1H), 4.07(s, 1H), 4.50 (s, 2H), 5.26 (d, 1H, J=7.5 Hz), 7.30 (m, 5H)

(2R,3S)-4-benzyloxy-2-N-tert-butoxycarbonylamino-1,3-bis(tert-butyldimethylsilyloxy)-butane(5)

t-Butyldimethylsilyl trifluoromethanesulfonate (4.4 mL, 19 mmol) and2,6-lutidine (3.0 mL) were added to a methylene chloride solution (100mL) containing a diol (4) (2.0 g, 6.4 mmol) while cooling on ice, andthe mixture was stirred for 30 minutes. A saturated ammonium chlorideaqueous solution was added to quench the reaction, extraction wasperformed with ether, and the organic layer was washed with 1Nhydrochloric acid and a saturated sodium chloride aqueous solution anddried over magnesium sulfate. The solvent was distilled off and theresulting residue was purified by silica gel column chromatography(ether/hexane=1/3) to obtain 2.96 g of the title compound (86%) and 295mg of a monosilyl compound (11%).

Oily: [α]_(D) −7.7°(c1.71, CHCl₃); ¹H NMR (CDCl₃, 400 MHz); δ−0.01 (s,3H), 0.00 (s, 6H), 0.02 (s, 3H), 0.82 (s, 9H), 0.83 (s, 9H), 1.45 (s,9H), 3.39 (m, 3H), 3.56 (dd, 1H, J=5.0, 10.0 Hz), 3.70 (m, 1H), 4.12(dt, 1H, J=1.5, 6.0 Hz), 4.44 (s, 2H), 4.71 (d, 1H, J=9.0 Hz), 7.20 (m,1H), 7.28 (m, 4H).

(2S,3R)-3-tert-Butoxycarbonylamino-4-tert-butyldimethylsilyloxy-2-hydroxybutyricacid methyl ester (6)

After dissolving 2.96 g of a silyl-protected compound (5) (5.48 mmol) inmethanol (100 mL), palladium black (200 mg) was added and the mixturewas stirred for 3 hours under a hydrogen atmosphere. The catalyst wasfiltered off, the solvent was distilled off, and then the residue wasdissolved in 100 mL of methylene chloride. A mixture of oxalyl chloride(952 μL, 11 mmol) and DMSO (1.17 mL, 16.4 mmol) prepared in methylenechloride at −78° C. was added thereto at −78° C., and the reactionmixture was stirred at −78° C. for 10 minutes and at −50° C. for onehour. After adding triethylamine (3 mL, 22 mmol) to the reaction mixtureat −50° C., the temperature was raised to 0° C. and stirring wascontained for 5 minutes. A saturated ammonium chloride aqueous solutionwas added to quench the reaction, extraction was performed with ether,and the organic layer was washed with 1N hydrochloric acid and asaturated sodium chloride aqueous solution and dried over magnesiumsulfate. The residue obtained by distilling off the solvent wasdissolved in 50 mL of acetone, and Jones reagent was added at 0° C.until the solution turned a brown color. 2-Propanol was added to quenchthe reaction, and extraction was performed with ether. Diazomethane wasadded to the organic layer for methyl esterification, and the solutionwas dried over magnesium sulfate. The solvent was distilled off, theobtained residue was dissolved in 100 mL of THF, and a THF solution (1N, 11 mL) containing tetra-n-butylammonium fluoride was added. Ethylacetate was added for extraction, and the organic layer was washed witha 5% citric acid aqueous solution. The organic layer was then dried overmagnesium sulfate, and the lactone and diol mixture obtained bydistilling off the solvent was dissolved in methanol, after which acatalytic amount of acidic resin (Amberlyst 15E) was added and themixture was stirred for 16 hours. The catalyst was filtered off, thesolvent was distilled off, the obtained residue was dissolved in DMF,and then t-butyldimethylsilyl chloride (828 mg, 5.5 mmol) and imidazole(748 mg, 11 mmol) were added and the mixture was stirred at roomtemperature for 3 hours. After adding methanol to quench the reactionand performing extraction with ether, the organic layer was washed with1N hydrochloric acid and a saturated sodium chloride aqueous solutionand dried over magnesium sulfate. The solvent was distilled off and theresulting residue was purified by silica gel column chromatography(ether/hexane=1/3) to obtain 1.25 g of the title compound (7 steps,63%).

Oily: [α]_(D) +10.7° (c1.50, CHCl₃); ¹H NMR (CDCl₃, 400 MHz); δ0.03 (s,6H), 0.90 (s, 9H), 1.44 (s, 9H), 3.27 (d, 1H, J=4.0 Hz), 3.64 (dd, 1H,J=7.5 Hz, 9.5 Hz), 3.73 (dd, 1H, J=5.0, 9.5 Hz), 3.79 (s, 3H), 4.09 (m,1H), 4.46 (m, 1H), 4.87 (d, 1H, J=9.0 Hz).

(2S,3R)-3-N-tert-Butoxycarbonylamino-4-tert-butyldimethylsilyloxy-2-(3-nitrobenzyl)oxybutyricacid methyl ester (7)

After adding sodium hydride (116 mg, 2.90 mmol) andtetra-n-butylammonium iodide (213 mg, 0.60 mmol) to 5 mL of a DMFsolution containing a 2-OH compound (6) (700 mg, 1.93 mmol) at −20° C.,3-nitrobenzyl bromide (625 mg, 2.90 mmol) was further added thereto andthe mixture was stirred at −20° C. for 30 minutes and at 0° C. for 30minutes. A 5% citric acid aqueous solution was added to quench thereaction, extraction was performed with ether, and the organic layer wasdried over magnesium sulfate. The solvent was distilled off and theresulting residue was purified by silica gel column chromatography(ether/hexane=1/3) to obtain 759 mg of the title compound (79%).

Oily: [α]_(D) −8.7° (c 1.94, CHCl₃); ¹H NMR (CDCl₃, 400 MHz); δ0.03 (s,3H), 0.06 (s, 3H), 0.86 (s, 9H), 1.40 (s, 9H), 3.57 (dd, 1H, J=9.5, 9.5Hz), 3.69 (dd, 1H, J=5.0, 9.5 Hz), 3.77 (s, 3H), 4.18 (m, 1H), 4.39 (d,1H, J=2.0 Hz), 4.50 (d, 1H, J=12.0 Hz), 4.86 (d, 1H, J=10.0 Hz), 4.88(d, 1H, J=12.0 Hz), 7.53 (dd, 1H, J=8.0, 8.0 Hz), 7.71 (d, 1H, J=8.0Hz), 8.16 (m, 1H), 8.22 (s, 1H).

(2S,3R)-2-[3-(3-N-Benzyloxycarbonylaminopropionylamino)benzyloxy]-3-N-tert-butoxycarbonylamino-4-tert-butyldimethylsilyloxybutyricacid methyl ester (8)

A catalytic amount of palladium carbon (10%) was added to 30 mL of amethanol solution containing a nitrobenzyl compound (7) (759 mg, 1.52mmol), and the mixture was stirred for 3 hours under a hydrogenatmosphere. The catalyst was filtered off, and the filtrate wasconcentrated under reduced pressure. The residue was dissolved in 50 mLof methylene chloride, and then N-benzyloxycarbonyl-β-alanine (439 mg, 2mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(406 mg, 2 mmol) were added and the mixture was stirred at roomtemperature for 30 minutes. Ether and water were added for extraction,and the organic layer was washed with a 5% citric acid aqueous solution,water, a saturated sodium bicarbonate aqueous solution and water in thatorder. The organic layer was dried over magnesium sulfate, the solventwas distilled off and the resulting residue was purified by silica gelcolumn chromatography (ether/hexane=3/1) to obtain 956 mg of the titlecompound (2 steps, 94%).

Oily: [α]_(D) −5.3° (c1.68, CHCl₃); ¹H NMR (CDCl₃, 400 MHz); δ0.03 (s,3H), 0.06 (s, 3H), 0.89 (s, 9H), 1.40 (s, 9H), 2.58 (m, 2H), 3.54 (m,3H), 3.64 (dd, 1H, J=5.0, 9.5 Hz), 3.74 (s, 3H), 4.13 (m, 1H), 4.32 (brs, 1H), 4.36 (d, 1H, J=11.5 Hz), 4.69 (d, 1H, J=11.5 Hz), 4.98 (d, 1H,J=10.0 Hz), 5.08 (m, 2H), 5.66 (br s, 1H), 7.08 (d, 1H, J=7.0 Hz), 7.30(m, 6H), 7.48 (m, 2H), 7.96 (s, 1H).

(2S,3S)-3-[3-(3-N-Benzyloxycarbonylaminopropionylamino)benzyloxy]-2-N-tert-butoxycarbonyl-aspartic acid δ-methyl ester (9)

A catalytic amount of DL-camphorsulfonic acid was added to 10 mL of amethanol solution containing compound (8) (777 mg, 1.15 mmol) and themixture was stirred for 5 hours. Ether and water were added forextraction, and the organic layer was dried over magnesium sulfate. Theresidue obtained by distilling off the solvent was dissolved in 5 mL ofacetone, and Jones reagent was added at 0° C. until the solutionbrownness no longer disappeared. 2-Propanol was added to quench thereaction, and extraction was performed with ether. After extraction intoan aqueous layer with a saturated sodium bicarbonate aqueous solution,the aqueous layer was adjusted to pH 2 with 2N hydrochloric acid, andextraction was again performed into an organic layer with ethyl acetate.The organic layer was dried over magnesium sulfate, and the residueobtained by distilling off the solvent was purified by silica gel columnchromatography (methanol/chloroform=1/19) to obtain 210 mg of the titlecompound (2 steps, 32%).

Oily: [α]_(D) −52.5° (c0.46, CHCl₃); ¹H NMR (CDCl₃, 400 MHz); δ1.40 (s,9H), 2.53 (m, 2H), 3.48 (m, 2H), 3.77 (s, 3H), 4.29 (d, 1H, J=12.5 Hz),4.45 (d, 1H, J=2.5 Hz), 4.81 (m, 2H), 5.07 (s, 2H), 5.48 (d, 1H, J=9.5Hz), 5.83 (m, 1H), 6.96 (d, 1H, J=7.0 Hz), 7.17-7.36 (m, 8H), 8.44 (brs, 1H).

(2S,3S)-3-[3-(3-aminopropionylamino)benzyloxy]aspartic acid (AA-TBOA)

After adding 1.5 mL of a 1N sodium hydroxide aqueous solution to 1 mL ofa methanol solution of Compound (9) (190 mg, 0.33 mmol), the mixture wasstirred while cooling on ice for one hour and at room temperature for 16hours. After adding 1 mL of 2N hydrochloric acid, extraction wasperformed with ethyl acetate, and the organic layer was washed with asaturated sodium chloride aqueous solution and dried over magnesiumsulfate. The residue obtained by distilling off the solvent wasdissolved in methanol (5 mL), after which catalytic amounts ofhydrochloric acid and palladium black were added and the mixture wasstirred under a hydrogen atmosphere for 3 hours. The catalyst wasfiltered off, the residue obtained by concentrating the filtrate wasdissolved in 2 mL of methylene chloride, 1 mL of trifluoroacetic acidwas added and the mixture was stirred for 15 minutes. The solvent wasdistilled off, the residue was subjected to Dowex 50×100 columnchromatography and washed with water, and then elution was performedwith 1N ammonia water. Lyophilization was repeated to obtain the titlecompound (97 mg, 91%).

Amorphous: [α]_(D) −25.0° (c 0.88, H₂O); ¹H NMR (D₂O, 400 MHz); δ2.74(t, 2H, J=6.5 Hz), 3.18 (t, 2H, J=6.5 Hz), 3.69 (s, 1H), 4.24 (s, 1H),4.42 (d, 1H, J=12.0 Hz), 4.71 (d, 1H, J=12.0 Hz), 7.22 (d, 1H, J=7.0Hz), 7.36 (d, 1H, J=7.0 Hz), 7.41 (dd, J=7.0 Hz, 7.5 Hz), 7.50 (d, 1H,J=7.5 Hz).

(2S,3S)-3-[3-(3-N-biotinylaminopropionylamino)benzyloxy]-2-N-tert-butoxycarbonyl-asparticacid δ-methyl ester (10)

A catalytic amount of palladium carbon (10%) was added to 5 mL of amethanol solution containing compound (8) (107 mg, 0.20 mmol), and themixture was stirred for 3 hours under a hydrogen atmosphere. Thecatalyst was filtered off, the residue obtained by concentrating thefiltrate was dissolved in 2 mL of methylene chloride, and then biotin,pentafluorophenyl ester (91 mg, 0.22 mmol) and triethylamine (30 μL,0.22 mmol) were added and the mixture was stirred at room temperaturefor 30 minutes. The reaction solution was diluted with ether, and washedwith 1N hydrochloric acid and water. The organic layer was dried overmagnesium sulfate.

The residue obtained by distilling off the solvent was purified bysilica gel column chromatography (methanol/chloroform=1/10) to obtain 55mg of the title compound (2 steps, 46%).

Oily: [α]_(D) −26.8° (c 0.58, MeOH) ¹H NMR (CDCl₃, 400 MHz); δ1.40 (s,9H), 1.5-1.8 (m, 6H), 2.26 (m, 2H), 2.66 (m, 2H), 2.77 (m, 3H), 2.89(dd, 1H, J=4.5, 13.0 Hz), 3.13 (m, 1H), 3.46 (m, 1H), 3.68 (m, 1H), 3.75(s, 3H), 4.34 (m, 1H), 4.42 (d, 1H, J=13.0 Hz), 4.44 (d, 1H, J=2.0 Hz),4.51 (m, 1H), 4.80 (dd, 1H, J=2.0, 9.5 Hz), 4.91 (d, 1H, J=13.0 Hz),5.49 (d, 1H, J=9.5 Hz), 6.52 (m, 1H), 6.61 (s, 1H), 6.66 (s, 1H), 6.86(d, 1H, J=7.5 Hz), 7.02 (s, 1H), 8.04 (d, 1H, J=8.0 Hz), 8.87 (s, 1H).

(2S,3S)-3-[3-(3-N-biotinylaminopropionylamino)benzyloxy] aspartic acid(Bio-AA-TBOA)

After adding 66 μL of a 1N sodium hydroxide aqueous solution to 1 mL ofa methanol solution containing compound (10) (13 mg, 0.22 mmol), themixture was stirred at room temperature for 18 hours. The reactionsolution was adjusted to pH 1 with 2N hydrochloric acid, and extractionwas performed with ethyl acetate. The organic layer was dried overmagnesium sulfate, and the residue obtained by distilling off thesolvent was dissolved in 1 mL of chloroform prior to adding 1 mL oftrifluoroacetic acid and stirring for 15 minutes. The solvent wasdistilled off, the residue was subjected to Dowex 50×100 columnchromatography and washed with water, and then elution was performedwith 1N ammonia water. Lyophilization was repeated to obtain 7.9 mg ofthe title compound (72%).

Amorphous: [α]_(D) +6.1° (c 0.32, 50% DMSO-H₂O); ¹H NMR (D₂O, 400 MHz);δ1.15 (m, 2H), 1.32 (m, 1H), 1.45 (m, 3H), 2.53 (dd, 1H, J=4.0, 12.5Hz), 2.54 (m, 2H), 2.66 (dd, 1H, J=5.0, 12.5 Hz), 2.81 (m, 1H), 3.44 (m,2H), 3.48 (d, 1H, J=4.5 Hz), 3.90 (dd, 1H, J=1.0, 2.0 Hz), 3.94 (dd, 1H,J=4.5, 8.0 Hz), 4.24 (m, 2H), 4.37 (d, 1H, J=12 Hz), 4.60 (d, 1H, J=12Hz), 7.09 (d, 1H, J=7.5 Hz), 7.22 (s, 1H), 7.29 (dd, 1H, J=7.5, 7.5 Hz),7.38 (d, 1H, J=7.5 Hz).

(2S,3R)-2-[3-(3-N-propionylaminopropionylamino)benzyloxy]-3-N-tert-butoxy-carbonylamino-4-tert-butyldimethylsilyloxybutyricacid methyl ester (11)

A catalytic amount of palladium carbon (10%) was added to 10 mL of amethanol solution containing compound (8) (110 mg, 0.16 mmol), and themixture was stirred for 3 hours under a hydrogen atmosphere. Thecatalyst was filtered off, and the filtrate was concentrated underreduced pressure. The residue was dissolved in 10 mL of methylenechloride, and then triethylamine (45 μL, 0.32 mmol) and propionylchloride (16 μL, 0.19 mmol) were added while cooling on ice and themixture was stirred for 15 minutes. Ether and water were added forextraction, and the organic layer was washed with a saturated sodiumbicarbonate aqueous solution, a 5% citric acid aqueous solution andwater in that order. The organic layer was dried over magnesium sulfate,the solvent was distilled off and the resulting residue was purified bysilica gel column chromatography (ether/hexane=3/1) to obtain 75 mg ofthe title compound (2 steps, 79%).

Oily: ¹H NMR (CDCl₃, 400 MHz); δ0.03 (s, 3H), 0.05 (s, 3H), 0.87 (s,9H), 1.12 (t, 3H, J=7.5 Hz), 1.40 (s, 9H), 2.19 (q, 2H, J=7.5 Hz), 2.62(t, 2H, J=5.5 Hz), 3.58 (m, 4H), 3.75 (s, 3H), 4.11 (m, 1H), 4.32 (d,1H, J=1.5 Hz), 4.38 (d, 1H, J=11.5 Hz), 4.73 (d, 1H, J=11.5 Hz), 4.98(d, 1 H, J=9.5 Hz), 6.44 (t, 1H, J=5.5 Hz), 7.09 (d, 1H, J=7.5 Hz), 7.28(m, 1H), 7.51 (m, 2H), 8.38 (s, 1H).

(2S,3S)-3-[3-(3-N-propionylaminopropionylamino)benzyloxy]aspartic acid(Pr-AA-TBOA)

A catalytic amount of DL-camphorsulfonic acid was added to 10 mL of amethanol solution containing compound (11) (57 mg, 0.096 mmol) and themixture was stirred for 3 hours. Ether and water were added forextraction, and the organic layer was dried over magnesium sulfate. Thesolvent was distilled off, the resulting residue was dissolved in 5 mLof acetone, and Jones reagent was added at 0° C. until the solutionbrownness no longer disappeared. 2-Propanol was added to quench thereaction, and extraction was performed with ether. After extraction intoan aqueous layer with a saturated sodium bicarbonate aqueous solution,the aqueous layer was adjusted to pH 2 with 2N hydrochloric acid, andextraction was again performed into an organic layer with ethyl acetate.The organic layer was dried over magnesium sulfate and the residueobtained by distilling off the solvent was dissolved in 1 mL ofmethanol, after which 1 mL of a 1N sodium hydroxide aqueous solution wasadded and the mixture was stirred at room temperature for 18 hours. Thereaction solution was adjusted to pH 1 with 2N hydrochloric acid, andextraction was performed with ethyl acetate. The organic layer was driedover magnesium sulfate, and the residue obtained by distilling off thesolvent was dissolved in 1 mL of chloroform prior to adding 1 mL oftrifluoroacetic acid and stirring for 15 minutes. The solvent wasdistilled off, the residue was subjected to Dowex 50×100 columnchromatography and washed with water, and then elution was performedwith 1N ammonia water. Lyophilization was repeated to obtain 14 mg ofthe title compound (5 steps, 39%).

Amorphous: [α]_(D) −17.3° (c 0.71, H₂O); ¹H NMR (D₂O, 400 MHz); δ1.11(t, 3H, J=7.5 Hz), 2.27 (q, 2H, J=7.5 Hz), 2.66 (t, 2H, J=6.5 Hz), 3.57(t, 2H, J=6.5 Hz), 3.68 (s, 1H), 4.24 (s, 1H), 4.44 (d, 1H, J=12.0 Hz),4.72 (d, 1H, J=12.0 Hz), 7.24 (d, 1H, J=7.5 Hz), 7.32 (s, 1H), 7.43 (t,1H, J=7.5 Hz), 7.51 (d, 1H, J=7.5 Hz).

As compounds without β-alanine, there were also synthesized A-TBOA,Bio-A-TBOA, Pr-A-TBOA, Piv-A-TBOA, PhAcA-TBOA, cHexcA-TBOA, BzA-TBOA,o-MeO-BzA-TBOA, m-MeO-BzA-TBOA, p-MeO-BzA-TBOA, diMeO-BzA-TBOA,tBu-BzA-TBOA, Ph-BzA-TBOA, CN-BzA-TBOA, NO₂-BzA-TBOA, F-BzA-TBOA,OCF₃-BzA-TBOA, CF₃-BzA-TBOA, OHex-BzA-TBOA and Hep-BzA-TBOA, by the samemethod according to Scheme 3.

(2S,3S)-3-(3-aminobenzyloxy)aspartic acid (A-TBOA)

Amorphous: ¹H NMR (D₂O, 400 MHz); δ3.86 (d, 1H, J=2.0 Hz), 4.19 (d, 1H,J=2.0 Hz), 4.26 (d, 1H, J=11.6 Hz), 4.52 (d, 1H, J=11.6 Hz), 6.70 (m,3H), 7.10 (t, 1H, J=7.6 Hz).

(2S,3S)-3-[3-(N-biotinylamino)benzyloxy]aspartic acid (Bio-A-TBOA)

Amorphous: [α]_(D) +31.1° (c 0.35, H₂O); δ1.53 (m, 2H), 1.68 (m, 1H),1.79 (m, 3H), 2.49 (t, 2H, J=7.8 Hz), 2.82 (d, 1H, J=13.0 Hz), 3.04 (dd,1H, J=5.0, 13.0 Hz), 3.40 (m, 1H), 3.70 (s, 1H), 4.26 (s, 1H), 4.44 (d,1H, J=12.0 Hz), 4.47 (dd, 1H, J=4.5, 8.0 Hz), 4.67 (dd, 1H, J=5.0, 7.5Hz), 4.73 (d, 1H, J=12.0 Hz), 7.25 (d, 1H, J=7.5 Hz), 7.32 (s, 1H), 7.44(t, 1H, J=7.5 Hz), 7.53 (d, 1H, J=7.5 Hz).

(2S,3S)-3-[3-(N-propionylamino)benzyloxy]aspartic acid (Pr-A-TBOA)

Amorphous: ¹H NMR (D₂O, 400 MHz); δ1.07 (t, 3H, J=7.5 Hz), 2.30 (q, 2H,J=7.5 Hz), 3.70 (s, 1H), 4.14 (s, 1H), 4.31 (d, 1H, J=12.0 Hz), 4.58 (d,1H, J=12.0 Hz), 7.08 (d, 1H, J=7.5 Hz), 7.13 (s, 1H), 7.28 (d, 1H, J=7.5Hz), 7.34 (d, 1H, J=8.0 Hz).

(2S,3S)-3-[3-(N-pivaroylamino)benzyloxy]aspartic acid (Piv-A-TBOA)

Amorphous: [α]_(D) −27.7° (c 0.31, H₂O); ¹H NMR (D₂O, 400 MHz); δ1.20(s, 9H), 3.86 (dd, 1H, J=2.0, 2.5 Hz), 4.20 (dd, 1H, J=2.0, 2.5 Hz),4.34 (d, 1H, J=12 Hz), 4.57 (d, 1H, J=12 Hz), 7.10 (d, 1H, J=7.0 Hz),7.17 (s, 1H), 7.25 (m, 2H)

(2S,3S)-3-[3-(N-phenylacetylamino)benzyloxy]aspartic acid (PhAcA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 3.61 (s, 2H), 3.89 (d, 1H, J=8.5 Hz), 4.16 (d,1H, J=8.5 Hz), 4.43 (d, 1H, J=11.0 Hz), 4.76 (d, 1H, J=11.0 Hz), 7.14(d, 1H, J=7.0 Hz), 7.20-7.35 (m, 6H), 7.53 (d, 1H, J=7.0 Hz), 7.55 (s,1H).

(2S,3S)-3-[3-(N-cyclohexylcarbonylamino)benzyloxy]aspartic acid(cHexcA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 1.10-1.42 (m, 6H), 1.73 (m, 4H), 2.29 (m, 1H),4.27 (d, 1H, J=3.5 Hz), 4.46 (d, 1H, J=12.0 Hz), 4.49 (d, 1H, J=3.5 Hz),4.72 (d, 1H, J=12.0 Hz), 7.03 (d, 1H, J=7.5 Hz), 7.26 (t, 1H, J=7.5 Hz),7.46 (d, 1H, J=8.0 Hz), 7.55 (s, 1H).

(2S,3S)-3-[3-(N-benzoylamino)benzyloxy]aspartic acid (BzA-TBOA)

¹H-NMR (CD₃OD) δ: 4.46 (d, 1H, J=11.5 Hz), 4.59 (d, 1H, J=2.5 Hz), 4.72(d, 1H, J=2.5 Hz), 4.82 (d, 1H, J=11.5 Hz), 7.14 (d, 1H, J=7.5 Hz), 7.34(t, 1H, J=7.5 Hz), 7.50 (m, 2H), 7.57 (m, 1H), 7.63 (s, 1H), 7.67 (d,1H, J=7.5 Hz), 7.93 (m, 2H).

(2S ,3S)-3-[3-[N-(2-methoxybenzoyl)amino]benzyloxy]aspartic acid(o-MeO-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 3.86 (3H, s), 4.06 (d, 1H, J=4.0 Hz), 4.42 (d,1H, J=4.0 Hz), 4.48 (d, 1H, J=11.5 Hz), 4.72 (d, 1H, J=11.5 Hz), 7.05(t, 1H, J=7.5 Hz), 7.19 (d, 1H, J=7.5 Hz), 7.14 (d, 1H, J=7.5 Hz), 7.31(t, 1H, J=7.5 Hz), 7.49 (dt, 1H, J=1.5, 8.0 Hz), 7.59 (s, 1H), 7.60 (d,1H, J=7.5 Hz), 7.65 (dd, 1H, J=1.5, 7.5 Hz).

(2S,3S)-3-[3-[N-(3-methoxybenzoyl)amino]benzyloxy]aspartic acid(m-MeO-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 3.84 (s, 3H), 4.15 (d, 1H, J=4.5 Hz), 4.39 (d,1H, J=4.5 Hz), 4.50 (d, 1H, J=9.5 Hz), 4.80 (d, 1H, J=9.5 Hz), 7.16 (m,2H), 7.33 (t, 1H, J=6.5 Hz), 7.44 t, 1H, J=6.5 Hz), 7.47 (m, 1H), 7.53(d, 1H, J=6.5 Hz), 7.67 (d, 1H, J=7.0 Hz), 7.74 (s, 1H).

(2S,3S)-3-[3-[N-(4-methoxybenzoyl)amino]benzyloxy]aspartic acid(p-MeO-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ:3.82 (s, 3H), 3.95 (d, 1H, J=7.5 Hz), 4.23 (d,1H, J=7.5 Hz), 4.48 (d, 1H, J=11.0 Hz), 4.80 (d, 1H, J=11.0 Hz), 7.04(d, 2H, J=9.0 Hz), 7.18 (d, 1H, J=7.5 Hz), 7.30 (t, 1H, J=7.5 Hz), 7.66(d, 1H, J=7.5 Hz), 7.72 (s, 1H), 7.94 (d, 2H, J=9.0 Hz).

(2S,3S)-3-[3-[N-(3,4-dimethoxybenzoyl)amino]benzyloxy]aspartic acid(diMeO-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 3.79 (s, 6H), 4.14 (d, 1H, J=4.5 Hz), 4.41 (d,1H, J=4.5 Hz), 4.48 (d, 1H, J=9.5 Hz), 4.71 (d, 1H, J=9.5 Hz), 7.04 (d,1H, J=8.5 Hz), 7.10 (d, 1H, J=7.5 Hz), 7.31 (t, 1H, J=6.5 Hz), 7.46 (d,1H, J=2.0 Hz), 7.55 (dd, 1H, J=2.5, 8.5 Hz), 7.58 (d, 2H, J=8.5 Hz).

(2S,3S)-3-[3-[N-(4-tert-butylbenzoyl)amino]benzyloxy]aspartic acid(tBu-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 1.33 (s, 9H), 4.05 (s, 1H), 7.08 (d, 1H, J=7.0Hz), 7.32 (t, 1H, J=7.5 Hz), 7.48 (s, 2H), 7.50 (d, 2H, J=7.5 Hz), 7.74(d, 2H, J=7.5 Hz).

(2S,3S)-3-[3-[N-(4-phenylbenzoyl)amino]benzyloxy]aspartic acid(Ph-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 3.92 (d, 1H, J=3.5 Hz), 4.38 (d, 1H, J=3.5 Hz),4.48 (d, 1H, J=12.0 Hz), 4.71 (d, 1H, J=12.0 Hz), 7.13 (d, 1H, J=8.0Hz), 7.33 (t, 1H, J=8.0 Hz), 7.39 (t, 1H, J=7.5 Hz), 7.48 (t, 2H, J=7.5Hz), 7.64 (m, 2H), 7.50 (d, 2H, J=8.0 Hz), 7.78 (d, 2H, J=8.0 Hz), 7.98(d, 2H, J=8.0 Hz)

(2S,3S)-3-[3-[N-(4-cyanobenzoyl)amino]benzyloxy]aspartic acid(CN-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 4.30 (d, 1H, J=3.5 Hz), 4.52 (d, 1H, J=3.5 Hz),4.54 (d, 1H, J=12.0 Hz), 4.79 (d, 1H, J=12.0 Hz), 7.16 (d, 1H, J=7.5Hz), 7.37 (t, 1H, J=7.5 Hz), 7.64 (d, 1H, J=8.0 Hz), 7.71 (s, 1H), 7.98(d, 2H, J=8.5 Hz), 8.07 (d, 2H, J=8.5 Hz).

(2S,3S)-3-[3-[N-(4-nitrobenzoyl)amino]benzyloxy]aspartic acid(NO₂-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 4.28 (d, 1H, J=3.5 Hz), 4.50 (d, 1H, J=3.5 Hz),4.52 (d, 1H, J=11.5 Hz), 4.76 (d, 1H, J=11.5 Hz), 7.14 (d, 1H, J=8.0Hz), 7.36 (t, 1H, J=8.0 Hz), 7.62 (d, 1H, J=8.0 Hz), 7.69 (s, 1H), 8.10(d, 2H, J=8.5 Hz), 8.32 (d, 2H, J=8.5 Hz).

(2S,3S)-3-[3-[N-(4-fluorobenzoyl)amino]benzyloxy]aspartic acid(F-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 4.30 (d, 1H, J=3.5 Hz), 4.52 (d, 1H, J=3.5 Hz),4.53 (d, 1H, J=11.5 Hz), 4.77 (d, 1H, J=11.5 Hz), 7.12 (d, 1H, J=7.5Hz), 7.33 (m, 3H), 7.63 (d, 1H, J=8.0 Hz), 7.70 (s, 1H), 7.99 (m, 2H).

(2S,3S)-3-[3-[N-(4-trifluoromethoxybenzoyl)amino]benzyloxy]aspartic acid(OCF₃-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 4.25 (d, 1H, J=3.5 Hz), 4.48 (d, 1H, J=3.5 Hz),4.50 (d, 1H, J=12.0 Hz), 4.65 (d, 1H, J=12.0 Hz), 7.14 (d, 1H, J=8.0Hz), 7.32 (t, 1H, J=8.0 Hz), 7.46 (d, 2H, J=8.5 Hz), 7.59 (s, 1H), 7.61(d, 1H, J=8.0 Hz), 7.99 (d, 2H, J=8.5 Hz).

(2S,3S)-3-[3-[N-(4-trifluoromethylbenzoyl)amino]benzyloxy]aspartic acid(CF₃-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 4.28 (d, 1H, J=3.5 Hz), 4.51 (d, 1H, J=3.5 Hz),4.53 (d, 1H, J=12.0 Hz), 4.77 (d, 1H, J=12.0 Hz), 7.14 (d, 1H, J=7.5Hz), 7.36 (t, 1H, J=7.5 Hz), 7.63 (d, 1H, J=8.0 Hz), 7.70 (s, 1H), 7.87(d, 2H, J=8.5 Hz), 8.08 (d, 2H, J=8.5 Hz).

(2S,3S)-3-[3-[N-(4-n-hexyloxybenzoyl)amino]benzyloxy]aspartic acid(OHex-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 0.81 (t, 3H, J=7.0 Hz), 1.24 (m, 6H), 1.35 (m,2H), 3.92 (d, 1H, J=4.0 Hz), 3.99 (t, 2H, J=6.5 Hz), 4.38 (d, 1H, J=4.0Hz), 4.46 (d, 1H, J=11.5 Hz), 4.68 (d, 1H, J=11.5 Hz), 6.98 (d, 2H,J=9.0 Hz), 7.09 (d, 1H, d, J=7.5 Hz), 7.30 (t, 1H, J=7.5 Hz), 7.57 (s,1H), 7.58 (d, 1H, J=7.5 Hz), 7.84 (d, 2H, J=9.0 Hz).

(2S,3S)-3-[3-[N-(4-n-heptylbenzoyl)amino]benzyloxy]aspartic acid(Hep-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 0.83 (t, 3H, J=6.5 Hz), 1.24 (m, 8H), 1.56 (m,2H), 2.62 (t, 2H, J=7.5 Hz), 3.81 (d, 1H, J=6.5 Hz), 4.28 (d, 1H, J=6.5Hz), 4.48 (d, 1H, J=11.0 Hz), 4.75 (d, 1H, J=11.0 Hz), 7.17 (d, 1H,J=7.5 Hz), 7.30 (m, 3H), 7.68 (m, 2H), 7.85 (d, 2H, J=7.5 Hz).

A para-substituted form (p-Pr-A-TBOA) was also synthesized by the samemethod.

(2S,3S)-3-[4-(N-propionylamino)benzyloxy]aspartic acid (p-Pr-A-TBOA)

Amorphous: ¹H NMR (50% D₂O/DMSO-d₆, 400 MHz); δ1.03 (t, 3H, J=7.5 Hz),2.27 (q, 2H, J=7.5 Hz), 4.20 (s, 1H), 4.43 (d, 1H, J=12.0 Hz), 4.60 (s,1H), 4.66 (d, 1H, J=12.0 Hz), 7.22 (d, 2H, J=7.0 Hz), 7.39 (m, 2H).

(2S,3S)-3-[3-(N-(4-(5-aminopentyloxy)benzoyl)amino)benzyloxy]asparticacid (A-PenO-BzA-TBOA)

¹H-NMR (D₂O) δ: 1.36 (tt, 2H, J=7.5 Hz), 1.58 (tt, 2H, J=7.5 Hz), 1.66(tt, 2H, J=7.5 Hz), 2.86 (t, 2H, J=7.5 Hz), 3.94 (t, 2H, J=6.0 Hz), 4.30(s, 1H), 4.44 (d, 1H, J=12.0 Hz), 4.52 (s, 1H), 4.68 (d, 1H, J=12.0 Hz),6.89 (d, 2H, J=7.5 Hz), 7.08 (d, 1H, J=7.0 Hz), 7.31 (m, 3H), 7.66 (d,2H, J=7.5 Hz).

(2S,3S)-3-[3-(N-(4-(5-biotynylaminopentyloxy)benzoyl)amino)benzyloxy]aspartic acid (BioA-PenO-BzA-TBOA)

¹H-NMR (DMSO-d₆, D₂O) δ: 1.20-1.65 (m, 12H), 1.71 (m, 2H), 2.04 (t, 2HJ=7.0 Hz), 2.56 (d, 1H, J=13.0 Hz), 2.78 (dd, 1H, J=4.5, 13.0 Hz), 3.05(m, 3H), 4.01 (t, 2H, J=5.5 Hz), 4.13 (m, 1H), 4.32 (m, 2H), 4.48 (d,1H, J=11.5 Hz), 4.71 (d, 1H, J=11.5 Hz), 7.01 (d, 2H, J=8.0 Hz), 7.13(d, 1H, J=7.0 Hz), 7.30 (t, 1H, J=7.5 Hz), 7.64 (m, 2H), 7.89 (d, 2H,J=8.0 Hz).

Activity Evaluation

A known method (Shimamoto, K. et al., Mol. Pharmacol. 53, 195-201, 1998;Bioorg. Med. Chem. Lett. 10, 2407-2410, 2000) was used to measure theinhibitory effect on uptake of [¹⁴C]glutamate by human EAAT2 and EAAT3stably expressed on MDCK (Madin-Darby Canine Kidney) cells ortransiently expressed on COS-1 cells. The glutamate uptake activity wasmeasured by adding 1 μM of L-[¹⁴C]glutamate and a prescribedconcentration of sample, incubating for 12 minutes and then lysating thecells, and using a liquid scintillator to determine the radioactivityuptake. The uptake was expressed as a percentage, with 100% being theuptake with no compound (buffer alone) and 0% being the uptake with asodium-free solution. The IC₅₀ values are shown in Table 1.

TABLE 1 EAAT2 EAAT3 TBOA  1.4 ± 0.12 μM  1.3 ± 0.11 μM A-TBOA  2.1 ± 0.1μM  7.9 ± 0.76 μM AA-TBOA   13 ± 1.1 μM   13 ± 1.1 μM Pr-A-TBOA  2.0 ±0.21 μM  7.9 ± 0.46 μM Piv-A-TBOA  1.0 ± 0.13 μM  4.1 ± 0.67 μMPr-AA-TBOA  6.0 ± 1.4 μM  7.6 ± 0.45 μM BioA-TBOA   38 ± 13 μM   25 ±2.4 μM Bio-AA-TBOA  6.3 ± 0.39 μM   10 ± 0.93 μM PhAcA-TBOA  143 ± 7.9nM   529 ± 32 nM CHexA-TBOA  244 ± 12 nM   288 ± 8.0 nM BzA-TBOA   55 ±5.0 nM   726 ± 98 nM cHexcA-TBOA  145 ± 2.2 nM  1000 ± 42 nMm-MeO-BzA-TBOA   37 ± 2.5 nM   356 ± 12 nM p-MeO-BzA-TBOA   12 ± 0.5 nM  266 ± 20 nM DiMeO-BzA-TBOA   49 ± 3.8 nM   917 ± 30 nM TBu-BzA-TBOA  25 ± 0.5 nM   182 ± 11 nM Ph-BzA-TBOA   21 ± 2.2 nM   34 ± 19 nMTBu-BzA-TBOA   36 ± 2.9 nM  1400 ± 99 nM NO₂-BzA-TBOA   14 ± 1.4 nM  278 ± 32 nM F-BzA-TBOA   22 ± 2.7 nM   473 ± 13 nM OCF₃-BzA-TBOA  7.0± 0.5 nM   128 ± 12 nM CF₃-BzA-TBOA  1.9 ± 0.10 nM   28 ± 1.8 nMOHex-BzA-TBOA  1.2 ± 0.04 nM   18 ± 1.6 nM p-Pr-A-TBOA  111 ± 25 μM*  295 ± 118 μM* A-PenO-BzA-TBOA  7.3 ± 1.1 μM  2.2 ± 0.20 μMBioA-PenO-BzA-TBOA  2.2 ± 0.52 μM  1.1 ± 0.06 μM The “*” indicatesresults obtained using COS-1 cells, and the other results were obtainedusing MDCK cells.

The results show that the inhibitory effect of the compounds of theinvention on uptake of [¹⁴C]glutamate by human EAAT2 and EAAT3 is atleast comparable to, or in some compounds, remarkably higher than thatof TBOA. Besides, it will be appreciated that the inhibitory effect ofsome compounds in table 1 is selective to EAAT2 over EAAT3.

Preparation of Affinity Column Ligand Preparatiom Example 1

A prescribed amount of CNBr-activated Sepharose 4B (AmershamBiosciences) is weighed on a glass filter. Washing and swelling arerepeated using 1 mM HCl. A compound of the invention (ligand) issolubilized in a coupling buffer (e.g., 0.1 M NaHCO₃ containing 0.5 MNaCl, pH 8.3). The ligand solution is mixed with the gel suspension fortwo hours at an ambient temperature or overnight at 4° C. Subsequently,the gell is introduced into a blocking agent such as 1 M ethanolamine or0.2 M glycine, pH 8.0. The gel is washed with the coupling buffer andacetate buffer (0.5 M NaCl, 0.1 M AcOH, pH 4) in that order to removeexcess ligand and blocking agent. The gel is stored at 4-8%.

Preparation Example 2

A prescribed amount of ECH Sepharose 4B (Amersham Biosciences) isweighed on a glass filter and washed with 0.5 M NaCl. A compound of theinvention (ligand) is solubilized in water, which is then adjusted at pH4.5. An aqueous solution of carbodiimide is prepared and the pH of thesolution is adjusted at pH 4.5. The swollen gel, the ligand solution andthe carbodiimide solution are mixed to allow to react at an ambienttemperature overnight. Excess ligand, urea derivatives and remainingligand are removed by washing. The gel is stored at 4-8° C.

Preparation Example 3

Immobilized avidin or streptavidin slury is poured into a column. Thepacked column is equilibrated with 5 column volumes of binding buffer(e.g., PBS). A biotinylated compound of the invention is applied to thecolumn and the column is incubated for 30 min. The column is washed with10 column volumes of binding buffer.

1. An L-threo-β-benzyloxyaspartate derivative having a substituent onthe benzene ring, represented by the following formula (1):

or a salt thereof, wherein R is hydrogen, a linear or branched loweraliphatic acyl group with the acyl portion optionally substituted, analicyclic acyl group, an aromatic acyl group with a substituent on thearomatic ring, an amino acid-derived group or a biotinderivative-derived group.
 2. A compound according to claim 1, wherein Ris hydrogen, an optionally thiol-substituted linear or branched loweraliphatic acyl group, an alicyclic acyl group, an aromatic acyl groupoptionally with a substituent on the aromatic ring, or a hydroxyl- orthiol-containing amino acid-derived group.
 3. A compound according toclaim 1, wherein R is acetyl, propionyl, n-butanoyl, sec-butanoyl,n-pentanoyl, pivaloyl, phenylacetyl, cyclohexylcarbonyl, benzoyl,substituted benzoyl, naphthoyl or pyridylcarbonyl.
 4. A compoundaccording to claim 1, wherein R is glycyl, alanyl, β-alanyl orcysteinyl.
 5. A compound according to claim 1, wherein R is biotinyl orbiotinyl-β-alanyl.
 6. A compound according to claim 3, wherein R is abenzoyl group substituted with one or more substituents selected fromthe group consisting of an optionally substituted alkoxy group, anoptionally substituted alkyl group, an optionally substituted arylgroup, a cyano group, a nitro group, and a halogen atom.
 7. A compoundaccording to claim 6, wherein said substituent on the benzoyl groupcontains an isotope.
 8. A method for producing anL-threo-nitrobenzylated (2S,3R) compound represented by the followingformula (7):

wherein Boc represents t-butoxycarbonyl and TBS representst-butyldimethylsilyl, wherein a compound of formula (6) as defined inclaim 6 is reacted with nitrobenzyl bromide in the presence of sodiumhydride and tetra-n-butylammonium iodide without altering thestereospecificity.
 9. The method of claim 8, wherein the yield of theL-threo-nitrobenzylated (2S,3R) compound represented by formula (7) isat least 70%.
 10. The method of claim 8, which further comprises a stepof producing a an L-threo-β-benzyloxyaspartate derivative having asubstituent on the benzene ring, represented by the following formula(1):

or a salt thereof, wherein R is hydrogen, a linear or branched loweraliphatic acyl group with the acyl portion optionally substituted, analicyclic acyl group, an aromatic acyl group with a substituent on thearomatic ring, an amino acid-derived group or a biotinderivative-derived group, by converting the NO₂ group on the benzenering of the compound of formula (7) to an NHR group, and removing anyprotecting groups.
 11. A compound represented by the following formula(7):

wherein Boc represents t-butoxycarbonyl and TBS representst-butyldimethylsilyl.
 12. A method for treating anL-glutamate-transporter-related neurodegenerative disease in a mammal,comprising administering to said mammal a pharmaceutical compositioncomprising an effective amount of a compound or a salt thereof asdefined in formula (1) in claim 1 , and a pharmaceutically acceptablecarrier, wherein the L-glutamate-transporter-related neurodegenerativedisease is selected from the group consisting of epilepsy, amyotrophiclateral sclerosis and Alzheimer's disease.
 13. A pharmaceuticalcomposition comprising an effective amount of a compound or a saltthereof as defined in formula (1) in claim 1, and a pharmaceuticallyacceptable carrier.
 14. A method for inhibiting anL-glutamate-transporter, comprising administering an effective amount ofa compound or a salt thereof as defined in claim
 12. 15. A method forinhibiting L-glutamate uptake, comprising administering an effectiveamount of a compound or a salt thereof as defined in claim 12.